The long term goal of this proposal is to understand the antigen processing mechanism by which endogenously synthesized or exogenous proteins yield precisely cleaved peptide/MHC class I complexes (pMHC I) on the cell surface. The expression of pMHC I is essential for the development and maintenance of the CD8+ cytotoxic T-cell repertoire and for immune responses specific for tumors, intracellular pathogens, allogeneic tissue grafts and self-tissues in autoimmunity. Failure to express pMHC I complexes is an important mechanism by which tumor cells and viral pathogens escape immune surveillance. We have developed novel cellular, biochemical and genetic techniques as well as model systems for analysis of naturally processed antigenic peptides. These methods allow the direct analysis of the final processed peptide products, and for the first time, their previously unknown proteolytic intermediates that are associated with cytoplasmic chaperones. Here we propose to test different hypotheses on how the early antigen processing steps shape the pool of naturally processed peptides available for presentation by MHC class I molecules. Furthermore, we will determine how exogenous antigens intersect with the endogenous antigen processing pathway and result in the display of pMHC I on the cell surface. We anticipate the answers will fill gaps in our understanding of the key factors that determine epitope selection, immunodominance and cross-presentation in the MHC class I antigen processing pathway. We anticipate that this research will improve our understanding of the mechanisms by which the immune system detects infected cells and tumors as well as self-tissues in autoimmunity.